The overall objective of the proposed research is to characterize the role of tissue specific cathepsins in extracellular matrix (ECM) protein degradation in cartilage. Previously, we and others identified cathepsins K and S as the predominant cysteine proteases in osteoclasts and macrophages, respectively, and characterized the extremely potent ECM- degrading activities of both cathepsins. We demonstrated that cathepsin K also is expressed in rheumatoid arthritis (RA) synovial fibroblasts and in mono- as well multi-nucleated cells of subchondral bone. Our working hypothesis is that cathepsins K and S are directly involved in cartilage degradation in arthritic joints. Therefore, in vitro and in vivo experiments will be undertaken to further characterize the biological functions of these cathepsins and to determine if these cathepsins cause excessive ECM degradation in tissue-degenerative pathologies such as RA. The expression of human cathepsins K and S will be localized in RA and non-RA joints and synovial fluid and correlated with the focal sites of cartilage erosion. Primary synovial fibroblast cultures from RA and non- RA specimens will be characterized with respect to the expression and secretion of both cathepsin activities. Substrate specificity studies will be undertaken with purified human cartilage proteins as well as with intact cartilage using recombinant cathepsins K and S. identified cleavage sites in ECM substrates will be used to produce specific neoepitope antibodies to identify cathepsin-released cleavage products in normal and diseased cartilage and synovial fluid. The extracellular processing of the cathepsin precursors will be assessed using known ECM-resident proteases. Furthermore, cooperative effects of cathepsin and MMP activities towards cartilage proteins will be determined. Ex-vivo cell models of cartilage erosion and antigen-induced arthritis mouse models using cathepsin K- deficient animals will be exploited to characterize the contribution of cathepsins K and S to the cartilage degeneration under non-inflammatory and inflammatory conditions. Altogether, these studies should determine the role of cathepsins K and S in ECM-protein degradation, and investigate novel strategies for the treatment of RA and other disorders with excessive ECM-degradation.